Project Summary: Dr. Craft established his independent research program in July 2005, focusing on tumor immunotherapy at Harbor-UCLA Medical Center with support from the LA Biomedical Research Institute. The environment is extremely supportive of new investigators making creative and entrepreneurial efforts in translational research amongst world leaders in related biomedical fields. The short term goals of this proposal are to define the mechanism of Toll-like receptor (TLR) enhancement of anti-melanoma vaccines and to optimize the vaccine strategies using a mouse model of melanoma. The long term goals are to provide a mechanistic foundation for the ongoing translational studies aimed at development of realistic targeted immunotherapies for humans with melanoma. These long term studies will be in collaboration with a multidisciplinary group of investigators and will be the subject of future R01 and other grant mechanisms. TLRs are a critical part of the immune system's ability to generate host defense to pathogens. Preliminary data suggests that the TLR7 agonist, imiquimod, is an effective adjuvant to immunization with recombinant Listeria monocytogenes (rLM) expressing melanoma antigens and leads to profound protection from melanoma. We hypothesize that stimulation of TLR7/8 leads to activation of dendritic cells (DCs) that in the context of vaccine induced CD8+ T cells leads to epitope spreading and tumor clearance. Aim 1 uses a mouse melanoma model to determine the molecular mechanism involved in TLR7/8 enhancement of an rLM-based anti-melanoma vaccine. Aim 2 will study the effects of TLR7/8 activation on DC based vaccines and optimize a combinatorial approach to melanoma immunotherapy using both DCs and rLM. Relevance: melanoma is a common cancer and a leading cause of loss of productive years. Many active trials for melanoma target tumor antigens with immunotherapy. This proposal is aimed at understanding the relationship between host immunity and self tolerance as it relates to tumor immunotherapy. These studies will improve our understanding of the mechanisms involved in anti-tumor immunity and will enhance the design of future immunotherapies for melanoma and other, less immunogenic cancers. This grant will provide the applicant with crucial funding during the initial independent years of his career and the results will be a stepping stone to development of realistic anti-melanoma vaccines and vaccine adjuvants.